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Trial registered on ANZCTR


Registration number
ACTRN12616000797471
Ethics application status
Approved
Date submitted
9/06/2016
Date registered
17/06/2016
Date last updated
6/12/2018
Date data sharing statement initially provided
6/12/2018
Type of registration
Prospectively registered

Titles & IDs
Public title
Probiotics and the EARly Life effects on intestinal bacteria and inflammation in children with Cystic Fibrosis (“PEARL-CF”)
Scientific title
Probiotics and the EARly Life effects on intestinal bacteria and inflammation in children with Cystic Fibrosis (“PEARL-CF”)
Secondary ID [1] 289412 0
None
Universal Trial Number (UTN)
Trial acronym
PEARL-CF
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Cystic fibrosis 299082 0
Condition category
Condition code
Human Genetics and Inherited Disorders 299113 299113 0 0
Cystic fibrosis
Inflammatory and Immune System 299165 299165 0 0
Other inflammatory or immune system disorders

Intervention/exposure
Study type
Interventional
Description of intervention(s) / exposure
This is a double-blind, randomised, placebo-controlled study in children comparing three cohorts:
(1) Subjects with CF taking probiotics,
(2) Subjects with CF taking a placebo, and
(3) Healthy non-CF controls (not on probiotics/placebo) matched to CF subjects (age and gender).

All subjects with CF will be randomised to either receive probiotic or placebo administered once daily for 12 months. Healthy controls do not receive probiotics or placebo.

Randomisation among CF subjects will be stratified according to the 2 age groups (0-3 and >3-6 years old) and performed as block randomization with a 1:1 allocation by an independent statistician. This age stratification is performed to evaluate the effects of probiotics when given at ages prior and after the age when the adult gut microbiota has normally established itself (~3 years old).

2g and 3g of probiotic or placebo powder will be administered daily to children 0-3 years and >3-6 years respectively. The powder allows for easy administration in infants and children, as it can be mixed into milk, juice, food (but avoiding hot drinks or food) or consumed directly off the spoon. Parents will be given a 2 months supply and instructed to store the probiotic appropriately at room temperature. A further 2 month supply will be provided at each stool sampling time point during the 12 months.

Selection of probiotic micro-organisms
A probiotic combination of Lactobacillus and Bifidobacterium spp will be used in this study.
Strains CFUs
Bifidobacterium lactis BI-04 3 Billion
Lactobacillus rhamnosus GG 2 Billion
Lactobacillus paracasei Lpc-37 1 Billion
Lactobacillus plantarum Lp-115 1 Billion
Lactobacillus rhamnosus HN001 500 Million
Lactobacillus rhamnosus Lr32 500 Million
Bifidobacterium animalis HN019 500 Million
Lactobacillus salivarius subsp. salivarius Ls-33 400 Million
Streptococcus thermophiles St21 400 Million
Bifidobacterium breve Bb-03 200 Million
Lactobacillus gasseri Lg 36 200 Million
Bifidobacterium longum BI-05 180 Million
Bifidobacterium infantis Bi-26 100 Million
Lactobacillus reuteri 1e1 100 Million
Lactobacillus bulgaricus Lb-64 100 Million

Excipient: Litesse Ultra (Polydextrose) 950 mg
Total (15 Strains) CFU per 1g: 10.2 Billion

For the control arm, at each stool collection time-point, the study investigator will enquire if participants are self-administering probiotics.
Intervention code [1] 294997 0
Treatment: Other
Intervention code [2] 295051 0
Prevention
Comparator / control treatment
All subjects with CF will be randomised to either receive probiotic or placebo administered once daily for 12 months. Healthy controls do not receive probiotics or placebo.

Selection of Placebo
The placebo will be composed of maltodextrin (a carbohydrate) and microcrystalline cellulose (MCC) at a ratio of 19:1 (i.e. for 2g dose – 1.9g maltodextrin and 0.1g MCC; for 3g dose 2.85g maltodextrin and 0.15g MCC). The placebo will be the identical to the probiotic in appearance, colour and solubility. Taste when mixed in a liquid is almost identical. The probiotic and placebo will be recommended to be given with products such as milk or apple puree (similar to instructions for pancreatic enzymes). Both the probiotic and placebo will be stored in identical sealed glass bottles which are only differentiated by a randomisation code.

Healthy non-CF controls (not on probiotics/placebo) matched to CF subjects (age and gender) will serve as a second comparator group.
Control group
Active

Outcomes
Primary outcome [1] 298586 0
Primary outcomes: Intestinal microbial profiles from stool samples will be measured by:
(i) Microbial richness expressed by the total number of operational taxonomic units at a species level
Timepoint [1] 298586 0
Baseline and 2 monthly for the first 12 months, and then 3 monthly for the next 12 months.
Primary outcome [2] 298650 0
Primary outcomes: Intestinal microbial profiles from stool samples will be measured by:
(ii) Microbial diversity using Shannon’s diversity index
Timepoint [2] 298650 0
Baseline and 2 monthly for the first 12 months, and then 3 monthly for the next 12 months.
Primary outcome [3] 298651 0
Primary outcomes: Intestinal microbial profiles from stool samples will be measured by:
(iii) Bray-Curtis dissimilarity
Timepoint [3] 298651 0
Baseline and 2 monthly for the first 12 months, and then 3 monthly for the next 12 months.
Secondary outcome [1] 324641 0
Intestinal inflammation as measured by stool calprotectin
Timepoint [1] 324641 0
Baseline and 2 monthly for the first 12 months, and then 3 monthly for the next 12 months.
Secondary outcome [2] 354696 0
Assessment of the faecal metabolome using stool supernatant measured by liquid chromatography mass spectrometry (LC-MS)
Timepoint [2] 354696 0
Baseline and 2 monthly for the first 12 months, and then 3 monthly for the next 12 months.
Secondary outcome [3] 354697 0
Assessment of the faecal proteome using stool supernatant analysed by LC-MS
Timepoint [3] 354697 0
Baseline and 2 monthly for the first 12 months, and then 3 monthly for the next 12 months.
Secondary outcome [4] 354698 0
Growth (height, weight and BMI) z-scores
Timepoint [4] 354698 0
Baseline and 2 monthly for the first 12 months, and then 3 monthly for the next 12 months.
Secondary outcome [5] 354699 0
Number of pulmonary exacerbations (Fuch’s criteria)
Timepoint [5] 354699 0
Baseline and 2 monthly for the first 12 months, and then 3 monthly for the next 12 months.
Secondary outcome [6] 354700 0
Number of therapeutic antibiotic courses.
This outcome is assessed using a study-specific questionnaire.
Timepoint [6] 354700 0
Baseline and 2 monthly for the first 12 months, and then 3 monthly for the next 12 months.
Secondary outcome [7] 354701 0
Number of hospitalisations.
This outcome is assess using a study-specific questionnaire.
Timepoint [7] 354701 0
Baseline and 2 monthly for the first 12 months, and then 3 monthly for the next 12 months.
Secondary outcome [8] 354702 0
(OPTIONAL) Lung function testing - using sedated infant lung function (0 up to 3 years) or awake multiple breath washout testing (2 years and older).
This outcome is assess using a study-specific questionnaire.
Timepoint [8] 354702 0
Baseline, 12 months and 24 months.
Secondary outcome [9] 354703 0
Quality of life outcomes will be measured using age-appropriate PedsQL questionnaires:
• ages 1 to 12 months – Infant Scales v1.0 (Parent report for ages 1-12 months) [Varni et al., The PedsQL Infant Scales: feasibility, internal consistency reliability, and validity in healthy and ill infants. Quality of life research, 2011;20:45-55]
• ages 13 to 24 months – Infant Scales v1.0 (Parent report for ages 13-24 months) [Varni et al., The PedsQL Infant Scales: feasibility, internal consistency reliability, and validity in healthy and ill infants. Quality of life research, 2011;20:45-55]
• ages 2 to 4 years – Gastrointestinal Symptoms Module v3.0 (Parent report for ages 2-4) [Varni et al., PedsQL gastrointestinal symptoms module: feasibility, reliability, and validity. JPGN. 2014;59:347-55; Varni et al., PedsQL gastrointestinal symptoms module item development: qualitative methods. JPGN. 2012;54:664-71]
• ages 5 to 7 years – Gastrointestinal Symptoms Module v3.0 (Parent report for ages 5-7) [Varni et al., PedsQL gastrointestinal symptoms module: feasibility, reliability, and validity. JPGN. 2014;59:347-55; Varni et al., PedsQL gastrointestinal symptoms module item development: qualitative methods. JPGN. 2012;54:664-71]
Timepoint [9] 354703 0
Baseline and 2 monthly for the first 12 months, and then 3 monthly for the next 12 months.

Eligibility
Key inclusion criteria
(1) All subjects with CF must fulfil the diagnostic criteria for CF (sweat chloride greater or equal to 60 mmol/L and/or 2 CF-causing mutations).

(2) Healthy controls include healthy full term infants (38-40 weeks gestation; >2.5kg birth weight), and who do not have any major non-CF disease (e.g. spina bifida) or gut disease (e.g. inflammatory bowel disease).
Minimum age
No limit
Maximum age
6 Years
Gender
Both males and females
Can healthy volunteers participate?
Yes
Key exclusion criteria
1) Patients with CF who have had previous intestinal surgery (e.g. meconium ileus) and/or have intestinal resection resulting in short gut syndrome.
(2) Any CF subject already on probiotics but who are not willing to stop supplementation for 3 months prior to randomization.
(3) Healthy controls on probiotics who are not willing to stop.

Study design
Purpose of the study
Treatment
Allocation to intervention
Randomised controlled trial
Procedure for enrolling a subject and allocating the treatment (allocation concealment procedures)
Allocation is performed by the Clinical Pharmacists at each site. Clinical Pharmacists have no role in determining subject eligibility or recruitment. Subject eligibility and recruitment is performed by a study investigator who is blinded to the randomisation code and allocation.
Methods used to generate the sequence in which subjects will be randomised (sequence generation)
Subjects with CF will be randomly assigned to either receive probiotic supplementation or placebo with a 1:1 allocation using block randomisation (blocks of 2, 4 and 6) stratified by age groups of 0-3 years and >3-6 years. A randomisation code for each study site will be created by an independent third party using an online computer generated code (https://www.sealedenvelope.com). The randomisation code will then be sent to the Clinical Pharmacists for the respective study sites. The randomisation code will not be shown to any study investigators.
Masking / blinding
Blinded (masking used)
Who is / are masked / blinded?
The people receiving the treatment/s
The people administering the treatment/s
The people assessing the outcomes
The people analysing the results/data
Intervention assignment
Parallel
Other design features
Phase
Not Applicable
Type of endpoint(s)
Efficacy
Statistical methods / analysis
Statistical analysis of all outcome measures will be temporal and cross-sectional and conducted according to intention-to-treat principles where applicable. Descriptive statistics will be performed for outcome parameters between groups according to normality of distribution. Diversity indices including the log10 number of OTUs, Shannon-Weaver Index and Bray-Curtis Dissimilarity will be calculated. Each diversity index will be included in a linear random-effects mixed model to compare temporal changes between the 2 and 3 groups (CF on probiotics and CF on placebo +/- healthy controls). Confounders including mode of delivery, feeding type in infancy and ethnicity will be analysed and treated as random effects if appropriate. Principle coordinate analysis (PCoA) (unweighted and weighted) and/or nonmetric multidimensional scaling (NMDS) plots will be constructed depending on the nature of the data. Relative abundances of bacterial taxa at each time point for each participant will be represented graphically using bar charts. The analysis of composition of microbiomes (ANCOM) [Mandal et al., Analysis of composition of microbiomes: a novel method for studying microbial composition. Microbial ecology in health and disease 2015,26:27663] in R will be utilised to detect statistical significant differences in mean microbial taxa between the 3 groups over time. The ANCOM package uses compositional log-ratios, makes no distributional assumptions and can be implemented in a linear model framework, whilst adjusting for covariates and longitudinal data.
Principle components analysis (PCA) will be performed for faecal proteins and metabolites at each time-point. Kruskal-Wallis tests will be performed to determine significantly different protein clusters and metabolites between the 2 and 3 groups. Bacterial taxa will be correlated with proteins and metabolites using a distance matrix (Bray-Curtis) and a Mantel test of Spearman or Pearson correlations depending on the normality of the data. Faecal calprotectin, growth parameters and lung function will be analysed using linear random-effects mixed models. The number of pulmonary exacerbations, antibiotic courses and hospitalisations will be compared using parametric or non-parametric t-tests between the two CF groups. R v3.3.1. (including the VEGAN and ANCOM packages68) Progenesis, Cytoscape and Scaffold will be used. For all analyses, a two-sided P-value of <0.05 will be considered statistically significant and when applicable, multiple testing will be accounted for using a false discovery rate correction (Benjamini and Hochberg method).

Sample Size Estimate
In our initial gut microbiota study (Nielson et al, 2016) we examined microbial communities within the gastrointestinal tract of children with and without CF across a range of childhood ages (0.87 to 17 years). The beta-diversity (Bray-Curtis dissimilarity) for CF and HC cohorts were 23.6 ± 7.5% and 26.9 ± 9.5% respectively. The required sample size for this study was calculated using the following conditions: (i) we assume that patients with CF who take probiotics over 12 months will achieve an improvement in the number of species, Shannon-Weaver index and Bray-Curtis dissimilarity, such that their values are increased by 75% of the difference between CF patients and healthy controls by the end of 12 months; (ii) we used estimates of the mean for each measure at 3 time points, 0, 6 and 12 months; (iii) relative group sizes of 1:1:2 for CF-probiotic, CF-placebo and healthy controls respectively; (iv) our estimates are calculated to detect a main effect only of groups; (v) type 1 error probability of 0.05 (two-tailed); (vi) desired power of 0.8; (vii) the GLIMMPSE Software (http://glimmpse.samplesizeshop.org/#/) was used.
The means used for each measure are presented below:

Richness (Log10 OTUs) S-W index B-C dissimilarity (%)
0m 6m 12m 0m 6m 12m 0m 6m 12m
HC 2.4 2.5 2.55 3.25 3.5 3.7 26.9 26.9 26.9
CF Probiotic 2.05 2.20 2.35 2.5 2.825 3.15 23.6 24.425 25.25
CF Placebo 2.05 2.10 2.15 2.5 2.55 2.6 23.6 23.6 23.6
B-C dissimilarity, Bray-Curtis dissimilarity; S-W index, Shannon-Weaver index.


Results from the sample size calculation are presented below:

Desired Power Actual Power Total SS Alpha Beta Scale Variability Scale Test Error Variance Corrected SD Total for Each CF Group
Richness 0.8 0.805 81 0.05 1 0.5 HLT 0.02 0.425 26
S-W Index 0.8 0.876 20 0.05 1 0.5 HLT 0.02 0.425 7
B-C dissimilarity 0.8 0.813 56 0.05 1 0.5 HLT 6 6 19
HLT, Hotelling Lawley Trace; SD, standard deviation; SS, sample size.

Based on the largest estimate present above (Richness), a minimum sample size of 26 probiotic, 26 placebo and 52 healthy subjects are required to reject the null hypothesis that the population means of the experimental and placebo-controlled groups are equal with probability (power) 0.8 and Type I error of 0.05. Assuming a total refusal and drop-out rate of 20%, 32 subjects for each CF cohort will be required, i.e. 32 subjects with CF on probiotics (0-3 year olds: n=16; >3-6 year olds: n=16), 32 CF subjects on placebo (0-3 year olds: n=16; >3-6 year olds: n=16), and 64 healthy controls (0-3 year olds: n=32; >3-6 year olds: n=32).

Recruitment
Recruitment status
Recruiting
Date of first participant enrolment
Anticipated
Actual
Date of last participant enrolment
Anticipated
Actual
Date of last data collection
Anticipated
Actual
Sample size
Target
Accrual to date
Final
Recruitment in Australia
Recruitment state(s)
NSW,QLD,VIC
Recruitment hospital [1] 5951 0
Sydney Children's Hospital - Randwick
Recruitment hospital [2] 5952 0
The Royal Childrens Hospital - Parkville
Recruitment hospital [3] 12665 0
The Children's Hospital at Westmead - Westmead
Recruitment hospital [4] 12666 0
Lady Cilento Children's Hospital - South Brisbane
Recruitment postcode(s) [1] 13376 0
2031 - Randwick
Recruitment postcode(s) [2] 13377 0
3052 - Parkville
Recruitment postcode(s) [3] 25087 0
2145 - Westmead
Recruitment postcode(s) [4] 25088 0
4101 - South Brisbane
Recruitment outside Australia
Country [1] 21099 0
New Zealand
State/province [1] 21099 0
Christchurch

Funding & Sponsors
Funding source category [1] 293784 0
Charities/Societies/Foundations
Name [1] 293784 0
Thrasher Research Fund
Address [1] 293784 0
68 S Main St, Suite 400,
Salt Lake City, UT 84101
Country [1] 293784 0
United States of America
Funding source category [2] 301389 0
Charities/Societies/Foundations
Name [2] 301389 0
Cystic Fibrosis Foundation
Address [2] 301389 0
Cystic Fibrosis Foundation

4550 Montgomery Ave.
Suite 1100 N
Bethesda, MD 20814
Country [2] 301389 0
United States of America
Primary sponsor type
Hospital
Name
Sydney Children's Hospitals Network
Address
High Street, Randwick 2031, New South Wales
Country
Australia
Secondary sponsor category [1] 292619 0
None
Name [1] 292619 0
Address [1] 292619 0
Country [1] 292619 0

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 295220 0
Sydney Children's Hospital Network HREC
Ethics committee address [1] 295220 0
Ethics committee country [1] 295220 0
Australia
Date submitted for ethics approval [1] 295220 0
03/05/2016
Approval date [1] 295220 0
22/06/2016
Ethics approval number [1] 295220 0
HREC/16/SCHN/167

Summary
Brief summary
The PEARL-CF study is a multi-center, double-blind, randomized, placebo-controlled trial comparing three cohorts:
(1) Children with cystic fibrosis (CF) taking a probiotic daily for 1 year,
(2) Children with CF taking a placebo daily for 1 year, and
(3) Healthy non-CF controls (HC) not on probiotics or placebo (age and gender matched).

The hypothesis is that:
(i) Probiotics restore the abnormal gut microbiota in children with CF, which in turn reduces intestinal inflammation.
We also hypothesize that when:
(ii) Probiotics are administered daily for 1 year, they will have clinical benefits for patients with CF.
(iii) Probiotics are administered in early life (0-3 years), the effects, even when ceased, are sustained compared to when probiotics are given after the gut microbiota has become established (~3 years old).
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 66582 0
Dr (Keith) Chee Y. Ooi
Address 66582 0
Sydney Children's Hospital Randwick
High Street, Randwick 2031, New South Wales
Country 66582 0
Australia
Phone 66582 0
+61293821752
Fax 66582 0
+61293821401
Email 66582 0
keith.ooi@unsw.edu.au
Contact person for public queries
Name 66583 0
Dr Michael Coffey
Address 66583 0
Sydney Children's Hospital Randwick
High Street, Randwick 2031, New South Wales
Country 66583 0
Australia
Phone 66583 0
+61404949508
Fax 66583 0
+61293821401
Email 66583 0
michael.coffey@unsw.edu.au
Contact person for scientific queries
Name 66584 0
Dr (Keith) Chee Y. Ooi
Address 66584 0
Sydney Children's Hospital Randwick
High Street, Randwick 2031, New South Wales
Country 66584 0
Australia
Phone 66584 0
+61293821752
Fax 66584 0
+61293821401
Email 66584 0
keith.ooi@unsw.edu.au

Data sharing statement
Will individual participant data (IPD) for this trial be available (including data dictionaries)?
Undecided
No/undecided IPD sharing reason/comment
Deidentified individual participant data (including sequencing data) will be made publically available ONLY if requested by the Journal when publishing the manuscript relevant to this work.
What supporting documents are/will be available?
No other documents available
Summary results
No Results